Recent studies suggested that the binding of a cardiac glycoside, ouabain, to the Na, K-ATPase, induces an alteration in the affinity of membrane phospholipids to calcium, sodium and potassium. Specific questions addressed by this proposal include the following. (a) How far into the lipid bilayer is the consequence of structural alterations in the Na, K-ATPase propagated? (b) To what extent is the process of information transfer modified by specific phospholipids, cholesterol and the degree of order/ disorder in the lipid bilayer? (c) To what extent does the mechanism for information transfer through the lipid bilayer, link the reactivity of the Na, K-ATPase and other membrane proteins? (d) What are the environmental conditions and membrane compositions necessary for protein-lipid-protein linkages in biological membranes? Techniques and approaches include: measurements of calcium binding versus pH, temperature and series of monovalent and divalent cations; induction of different conformational states of the Na, K-ATPase; changes in the ratio of phospholipids to Na, K-ATPase and alterations in the composition of the Na, K-ATPase preparations with respect to lipids; assessment of the interdependence that may exist between molecules of the Na, K-ATPase and between the Na, K-ATPase and other membrane systems such as the adenylate cyclase; and an assessment of how ouabain modifies the physical state of the lipid bilayer through the use of fluorescence probes, differential scanning and batch calorimetry and small particle electrophoresis of the Na, K-ATPase preparations to determine electrophoretic mobility of the vesicles under various conditions.